Project Summary This research project will test the hypothesis that, in the diabetic milieu, S100A9 induces the calcification potential of macrophage-derived extracellular vesicles (EV), precursors of microcalcifications, contributing to the biomechanical instability of the vulnerable atherosclerotic plaque. S100A9, a recently identified biomarker of vulnerable plaques, increases in the blood of patients with type 1 diabetes, is expressed by macrophages, and is a component of EV. Our published studies linked macrophages and calcification, and showed that macrophages can release EV with a high calcification potential. The present study will explore further the role of proinflammatory macrophages in vascular calcification in diabetes. Specific Aim 1 will test the hypothesis in vitro that diabetic milieu promotes macrophage activation and accelerates release and mineralization of S100A9?enriched EV. These experiments will involve innovative methods for detection of EV microcalcifications and macrophage phenotypes, including density dependent scanning electron microscopy (DDSEM) combined with elemental analysis, high-resolution microscopy, nanoparticle tracking analysis, 3D-hydrogel system, proteomics, single cell RNA sequencing, and network analyses. Specific Aim 2 will test the hypothesis in vivo that S100A9 mediates diabetes-induced microcalcifications in atherosclerotic plaques. We expect that i) genetic deletion of S100A9, ii) macrophage-targeted siRNA silencing of S100A9, and iii) bone marrow transplantation from S100A9-deficient mice will retard the progression of microcalcification and subsequent rupture, as determined by molecular imaging and comprehensive histopathological analyses. Specific Aim 3 will quantitatively evaluate the impact of microcalcification on the biomechanical instability of the atherosclerotic plaque, using mathematical modeling and finite element analysis. These complementary studies will advance the field by identifying the role of macrophage S100A9 in microcalcification. To facilitate clinical translation of mouse data, we will employ human primary macrophages and atherosclerotic plaque specimens from patients with diabetes. The findings from this project will help to develop much needed anti-calcification therapies.